Numerous physical effects of caffeine consumption have been previously described. However, the effects of caffeine on brain structure and CSF production have not been reported on, to date. The results of the present study showed that long-term consumption of caffeine could cause ventriculomegaly, which appears to be mediated, in part, by increased production of CSF.
In addition to disturbances in CSF dynamics, reduction in white matter (periventricular leukomalacia) can cause secondary ventriculomegaly . Periventricular leukomalacia can be caused by hypoxia, perinatal stress and sepsis. Previous studies have shown that hypoxia-induced ventriculomegaly is mediated by the A1 adenosine receptor, which is a major target molecule of caffeine in the brain [33, 35]. Hypoxia-induced ventriculomegaly was not observed in mice that were deficient in the A1 adenosine receptor (A1AR-/-). By contrast, activation of the A1 adenosine receptor, during the first 2 postnatal weeks, leads to white matter injury. Moreover, ventriculomegaly was also observed in mice lacking the enzyme, adenosine deaminase, which degrades adenosine . Although we cannot rule out the possibility of periventricular leukomalacia, caused by long-term consumption of caffeine, the radiologists did not find any of the signs of leukomalacia on the brain MRI of the caffeine-treated rats in the present study.
Different effects of short- and long-term caffeine exposure have been previously reported [11, 17]. The effect inversion of caffeine was also observed in the present study. The acute treatment with caffeine reduced CSF production; however, chronic treatment increased CSF production. Three findings from the present study support up-regulation of the A1 adenosine receptor as associated with the observed effect inversion of caffeine. First, the expression of the A1 receptor was up-regulated in the caffeine-treated rats. Second, A1 agonist treatment was associated with the development of ventriculomegaly. Third, the expression of Na+, K+-ATPase was increased in both the caffeine-treated and the A1 agonist-treated rats. Although this continues to be debated, many previous reports have demonstrated up-regulation of the A1 adenosine receptor in caffeine-treated rats [14, 36, 39].
However, other mechanisms might also contribute to the effect inversion of caffeine. Conley et al. showed that the concentration of adenosine in the blood, after long-term consumption of caffeine, was increased more than tenfold . This increase in the blood adenosine, after long-term consumption, might mediate the effect inversion of caffeine. However, the increase in the adenosine levels after long-term consumption of caffeine has never been replicated . Another possible mechanism is the involvement of the A2A receptor. Because the A2A receptor plays a critical role in the regulation of the cerebral arteries [42, 43], the increased CBF observed in the caffeine-treated rats might be mediated by the A2A receptor. Moreover, the A2A receptor agonist, CGS21680, also induced ventriculomegaly (Figure 5). However, in the present study, we did not find an increase in the expression of the A2A receptor in the caffeine-treated rats.
Recently, Yang et al showed that some aspects of the long-term consumption of caffeine can be replicated by genetic manipulation of the A1 and A2A adenosine receptors [41, 44]. Caffeine (0.3 g/l in drinking water for 7-10 days) and the A1R-A2AR double heterozygote genotype increased locomotor activity and decreased the heart rate without significantly influencing body temperature. Despite the similarities in phenotype, the effects of long-term consumption of caffeine cannot be explained only by the blocking of the adenosine receptors. For example the reduction in heart rate after long-term consumption of caffeine is not due to the acute block of the adenosine receptors because acute caffeine administration does not produce a fall in the heart rate. Moreover, induction of Na+, K+-ATPase with long-term consumption of caffeine, in the present study, also cannot be explained by the blocking of or reduction in the adenosine receptors; this is because A1 adenosine receptor agonist treatment (Figure 5) and A1 adenosine receptor transgenic mice have been reported to have increased expression of Na+, K+-ATPase .
Several receptor signaling pathways have been implicated in the regulation of CSF production. Vasopressin and angiotensin II have been shown to decrease CSF production by epithelial effects and changes in blood supply. Involvement of the cAMP/PKA pathways and intracellular calcium, in these receptor signaling pathways, remains controversial. The results of the present study suggest that the adenosine receptor(s) may not directly regulate CSF production. Acute treatment with an A1 agonist or an A1 antagonist did not significantly change CSF production; these findings suggest that the A1 adenosine receptor may not regulate CSF production directly (Figure 6). Acute treatment with the A2A agonist or antagonist increased or decreased CSF production, respectively (Figure 6). However, the direct effects of the A2A adenosine receptor on epithelial cells could not be confirmed, because it regulates cerebral blood flow [42, 43]. Therefore, the adenosine receptor appears to regulate CSF production by controlling the expression of Na+, K+-ATPase and cerebral blood flow. However, the acute and direct effects of the adenosine receptor on CSF production require further study.
Na+, K+-ATPase extrudes 3 Na+ in exchange for 2 K+ during the hydrolysis of 1 ATP molecule . This enzyme is located in the luminal surface of the choroid epithelial cells. By contrast, it is located in the basolateral surface of all other transporting epithelia, both secretory and absorptive. The luminal Na+ extrusion, by this enzyme, is the primary event and driving force of CSF production. Despite its critical role in CSF production, the regulation of its activity and expression has been poorly characterized. Na+, K+-ATPase is composed of α1, β1, β2 and phospholemman in the choroid plexus. In the present study, we showed that the A1 adenosine receptor regulates the expression of Na+, K+-ATPase in the choroid plexus; this finding is consistent with previous results showing increased expression of Na+, K+-ATPase in A1 adenosine receptor transgenic mice . However, it has not been determined whether adenosine receptor signaling can regulate the activity of the enzyme directly. On the other hand, phospholemman has been suggested to be a target for phosphorylation-activated CSF production  Indeed, phospholemman is activated by PKA and has also been shown to be involved in the conduction of the anion current induced by cell swelling. However, PKA-dependent regulation of the Na+, K+-ATPase in the choroid plexus requires further study.
It is unclear why the blood caffeine levels of rats with ventriculomegaly were 3 times higher than in the rats without ventriculomegaly. There was no difference found in the daily water uptake and therefore the dose of caffeine was not significantly different between the controls and caffeine treated groups, consistent with a previous report . Perhaps a difference in the metabolism of caffeine in the liver might explain these findings. The underlying mechanism needs to be investigated further.